Updated : Fri, May 5, 2023 @ 2:34 PM
How does a BHQ dye contribute to producing superior data from your assays?
In qPCR, a quencher is a type of molecule attached to an oligonucleotide that quenches fluorescence emitted by a fluorophore when excited by FRET (Förster Resonance Energy Transfer). Due to their polyaromatic-azo backbone, the Black Hole Quencher™ (BHQ™) dyes are true dark quenchers with no native emission. This allows the use of multiple reporter dyes with the same quencher, reducing signal cross-talk and helping to yield high-quality data from assays. Dark quenchers simplify reporter dye detection, which makes them compatible with a broad range of image analysis instruments. Substituting electron-donating and withdrawing groups on the aromatic rings produces a complete series of quenchers with broad absorption curves that span the visible spectrum.
BHQ dyes can be paired with all common reporter dyes to construct efficiently quenched qPCR probes for multiplexing assays. In addition to quenching by FRET, BHQ dyes have also been shown to efficiently quench fluorescence through static quenching via formation of a ground state complex with the reporter dye. This combination of FRET and static quenching by BHQ dyes enables greater sensitivity and avoids the residual background signal common to fluorescing quenchers such as TAMRA, or low signal to noise ratio.
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Did you know? Six out of seven of the World Health Organization’s COVID-19 testing protocols use BHQ dyes. |
How does a fluorescence quencher work?
The efficiency of fluorescence quenching is dependent on distance. If the reporter fluorophore and quencher are far apart, there is fluorescence; if the reporter and quencher are close together in space, fluorescence is suppressed. The reporter and quencher are placed at specific sites in an oligonucleotide in a way that a change in their distance will produce a maximal change in fluorescence and effectively signal the event being monitored (often hybridisation or nuclease activity). Along with comprising the specific sequence for binding to the target, the oligo acts as a flexible tether linking the fluorescent reporter and quencher.
The difference between BHQ dye and TAMRA
TAMRA dye is an effective quencher for fluorophores with emission maxima less than 560 nm. Dyes with longer wavelength emissions will not be effectively quenched by TAMRA. In addition, TAMRA has its own fluorescence emission at around 580 nm which complicates data analysis due to crosstalk between the channels. In contrast, BHQ dyes are true "dark" quenchers generating no fluorescent signal. Their use simplifies design, implementation and interpretation of qPCR assays.
Furthermore, BHQ dyes have broad absorption spanning 480-580 nm (BHQ-1), 559-670 nm (BHQ-2) and 620-730 nm (BHQ-3), to enable use of a large range of spectrally distinct reporter dyes in multiplexed assay designs. With some dye pairings, FRET quenching is supplemented by the static quenching mechanism. Specifically, hydrophobic and electrostatic interactions facilitate the association of BHQ dyes with certain reporters to form an intramolecular dimer, for enhanced quenching and improved signal to noise ratios. Thus, BHQ dyes may quench some fluorophores whose emission spectrum lies beyond the limits of BHQ absorption.
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Did you know? The top ten molecular diagnostic manufacturers leverage BHQ technology in their assays. |
Binding reporters and quenchers to form new non-fluorescent species
A new non-fluorescent species called an intramolecular dimer is formed when a reporter (such as FAM) and a quencher (such as BHQ-1) bind together. The biomolecule linking the reporter and quencher acts as a tether and enables intramolecular association between the reporter and quencher. Intramolecular dimers are useful for stabilising molecules and to help them maintain their structure.
Freedom of design
With BHQ dyes, it’s possible to synthesise your own oligos by buying them directly, either as CPG-bound or as phosphoramidites or esters to help develop your own probes. Alternatively, you can use BHQ probes developed by Biosearch Technologies based on the same ground-breaking dark quenching technology to jump start your assays.
A multitude of applications
Since the introduction of the BHQ dye technology in 2000, scientists have used them as the quencher of choice for qPCR probes and other fluorescence-quenched probe applications. Indeed, BHQ dyes have found many varied applications; from studies enabling simultaneous in vivo monitoring of insulin and glucose, to the detection of sepsis-causing bacteria, accurate quantification of genome edited crops and tracking the activation of an apoptotic signalling pathway. How will you use these versatile dyes?
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Did you know? Biosearch Technologies is the patent owner and inventor of BHQ, as well as CAL Fluor™, and Quasar dyes? |
Recommended reading:
Innovative applications for Black Hole Quencher dyes
Leveraging quenching efficiency of Black Hole Quencher dyes for PCR and beyond
